For much of the history of electron microscopy up until the 1990s, sheet film was the main image recording medium. In 1990, electronic detectors based on scintillators coupled to scientific semiconductor image sensors with either fused fiber or lens optics were introduced and began to replace film for most image recording applications. One of the main advantages of these sensors was for the applications of electron energy loss spectroscopy (EELS) and electron diffraction (ED) due the dramatically increased dynamic range of these scintillator-optics-semiconductor sensor. Both of these applications involve the use of a focused beam, with specimen information derived from electrons which are diverted from the main path either due to energy loss (subsequently made manifest with a bending magnet) or due to electron diffraction. The deflected beams can be several to many orders of magnitude weaker than the un-deflected beam as shown in FIGS. 1A, 1B, 2A and 2B. The graph in FIG. 1B shows that the diffraction spots in FIG. 1A have dynamic range of several orders of magnitude. FIGS. 2A and 2B show the dynamic range needed in EELS. Semiconductor sensors, typically CCDs for imaging and photodiode arrays and CCDs for spectroscopy, have significantly higher dynamic range than film, especially scientific sensors which were made with larger pixels. Dynamic ranges of up to 20,000 have been shown possible in single exposures with merged multiple exposures extending that limit even further. In addition, scintillators coupled optically to the sensor protect the sensor from the radiation damage effects that can results from either direct exposure of the sensor to the beam or from exposure to x-rays generated at the scintillator which can then travel to the sensor. In the case of lens coupling, the glass of the lens and the distance the lens allows to be created between scintillator and sensor confer the protective effect. In the case of fused fiber optic plates, the high density glass provides the x-ray protection. In both cases, the beam is stopped long before it can hit the sensor and cause damage directly. For both these reasons, the scintillator/optic/semiconductor sensor has replaced film in 100% of diffraction applications and made possible the parallel acquisition of spectra, which wasn't an option at all before these sensors made it possible.
FIG. 3A shows a prior art lens-coupled scintillator indirect detector having a scintillator 301, a glass prism 302, optical lenses 303, 304 and a CCD detector 305. FIG. 3B shows a prior art fused fiber-optic plate coupled indirect detector having a scintillator 311, an optical fiber bundle, 312 and a CCD detector 313.
Certain imaging applications, most notably cryo-electron microscopy of proteins and cellular cryo-tomography drove a need for a replacement of film which did not have a dynamic range requirement but rather a sensitivity and resolution requirement. In the last few years, a class of detectors has been developed using radiation hardened silicon active pixel detectors which is now successfully replacing film and extending the resolution limits attainable in structural biology. This technology is usually referred to as direct detection technology and cameras using this technology as direct detectors. In contradistinction to these detectors, the scintillator/optic/sensor detectors described above are now commonly referred to as indirect detectors.
Direct detectors make these improvements in structural biology resolution in a number of ways. First, silicon, being lighter than typical scintillator and optical materials, scatters the electron beam less giving a finer point spread function. Second, the directly detected incoming electron makes a much stronger signal. Third, it is possible to thin the device to allow the beam to pass through without additional noisy backscatter. While thinning is possible with lens coupled scintillators (see, for example, U.S. Pat. No. 5,517,033 all references cited herein are incorporated by reference), thinning comes with a severe loss of signal strength. FIG. 3C shows a prior art non-thinned (“bulk”) direct detector 320 and FIG. 3D shows a prior art back-thinned direct detector 330. In a direct detector there is no sensitivity penalty for thinning.
Finally, as a result of the first three benefits described above, (improved point spread function, higher signal strength and reduced backscatter from thinning), the signal from a direct detector can be processed to result in a counting mode analogous to that often used with a photomultiplier tube and with the same benefit. When an incoming electron is counted as a 1and added into a frame buffer, the variations in energy deposited by the incoming electron are stripped off and not summed as they would be in the case for a linear, integrating detector, whether direct or indirect. Because the acquired image no longer contains that variation in energy, a nearly noise-free acquisition is possible.
A second variant of counting is possible in which the position of entry of the electron is estimated to sub-pixel accuracy by centroiding the deposited energy. This method is commonly referred to a super-resolution. FIG. 4 shows the detection of a single incident electron with a super-resolution detector. FIG. 4A shows an electron landing on an arbitrary location within a pixel on a multipixel detection device. FIG. 4B shows scatter from incoming electrons in a localized region near the point of entry to the electron. FIG. 4c shows how the amount of scattered detected signal in nearby pixels is related to the location of the electron's entry point. FIG. 4d shows that selection of the pixel with the highest scattered signal locates the point of entry to the nearest pixel. FIG. 4e shows that finding the center of mass of the distribution of scattered charge allows location of the entry point to sub-pixel accuracy.
Counting and super-resolution dramatically improve the sensitivity performance as measured by detective quantum efficiency (DQE), the ratio of detected signal to noise ratio to incoming signal to noise ratio over the performance of a silicon direct detection imager.
FIG. 5 shows the sensitivity improvement summarized: of direct detection over indirect detection (A), of counting direct detection over just direct detection (B) and of the effect that super-resolution adds signal over the Nyquist frequency (C) of the physical pixel. Part of the DQE benefit comes from the additional benefit of counting of dramatically reducing detection of background noise. It is clear that indirect detectors have a serious disadvantage in terms of sensitivity. This is both in terms of DQE as shown in the graph in FIG. 5, but also in terms of the background noise. Both Electron energy loss spectroscopy (EELS) and electron diffraction (ED) have a strong need for high sensitivity and good background rejection for the weak parts of the signal.
Direct detectors (DD) have the drawback that the electron, while deposing signal energy in the pixel, will also damage it due to charge injected into insulators, as well as knock-on damage to the silicon crystal structure. This gives direct detectors a lifetime dose limit. While this dose limit has been dramatically increased by improvements to pixel design layout, reductions in feature sizes which reduce oxide thickness and thereby allow trapped electrons to diffuse out more readily, and thinning, which eliminates the energy deposited by backscatter, total lifetime dose is still limited to significantly lower levels than that of fiber-optically or lens-optically coupled scintillators. This fact would be severely limiting in applications like EELS and ED for which the undeflected beam is often many orders of magnitude higher than the low intensity part of the signal and would reduce the sensor lifetime, which is measured in years for cyro-electron microscopy, to hours.
For a DD detector to count electron arrival events they must be spatially and temporally separated on the detector. DQE is only modestly affected for 300 kV electrons at event densities up to about 0.025 per pixel (˜40 pixels per electron event). This is accomplished by an extreme speedup in frame-rate. A framerate of 400 fps as used on the Gatan K2 Counting direct detection camera allows a dose rate of 10 electrons per pixel per second at that event density. While that dose rate is adequate for low-dose imaging in cryo-microscopy for which it was developed, it is too low for use in higher-dose and in high-dynamic range applications such as EELS and ED as described in FIGS. 1 and 2. While it is conceivable that frame rates could be increased enough to handle medium dose ranges as shown in FIG. 2B, it is unrealistic to think that counting could be used for the high intensity parts of either the EELS or the ED signals. For the current generation of K2 counting direct detector, a commercial camera developed by Gatan, Inc. which uses the prior art detector arrangement of FIG. 3D in conjunction with extremely fast readout to separate the incoming electron events into different frames and a fast processor to count or centroid the electron events and sum them, the useable dose rate is 400 times lower in counting mode than in linear mode. FIG. 6 shows the sparsification by speed needed to allow counting. On the left, the actual signal generated by a sparse beam is shown, illustrating that each event covers multiple pixels, with varying size. Sparsification needs to be sufficient to prevent miscounting or poor centroiding due to overlap of scatter from one event onto another. The image on the right of FIG. 6 shows the results of counting the frame on the left. It also illustrates the extent to which the variability both in event intensity and in event size is reduced through the counting process.
Relevant patents in the field include U.S. Pat. No. 7,952,073 (“Bilhorn”) and U.S. Pat. No. 8,334,512 (“Luecken”). All references cited herein are incorporated by reference in their entirety.
The inadequacy of indirect detectors in resolution and sensitivity for dealing with the weak parts of EELS and ED signals and the inadequacy of direct detection to deal with the strong parts of EELS and ED signals coupled with the lack of any workaround in the prior art for acquiring both strong and weak signals simultaneous with high quality creates a need for a new solution.
Electron energy loss spectroscopy (EELS) and electron diffraction (ED) would stand to benefit significantly if the capability to read the weakest signals were added to existing capability. This is especially true for scanning transmission electron microscope spectrum imaging (STEM SI), for which a spectrum is taken at each of a N×M raster of scanned specimen pixels and used to derive elemental and electronic contrast images. See Gatan datasheet, “GIF Quantum”, published March 2014. This technique, which requires high speed to cover a reasonable number of pixels over a reasonable area of a specimen requires high sensitivity to very weak signals in the regions used for elemental contrast and yet still needs to be able to acquire and digitize the un-deflected beam for normalization. Similar applications are being developed for electron diffraction with STEM with similar requirements.
U.S. Pat. No. 8,334,512 is a patent in the field and discusses use of a fast detector positioned below the thinned imaging detector at some distance as a zero-loss beam position detector but due to the poor resolution associated with its position, cannot be used as a detector for low-loss spectroscopic information.
In addition, EELS and ED signals in their most general application can, and especially in these STEM applications, do vary rapidly in time, making serial illumination first of a low-sensitivity, high-robustness detector and second of a high-sensitivity low-robustness detector an impractical solution for the high dynamic range application.
Therefore there exists a need in the prior art for a technology which would allow simultaneous robust and high-quality imaging of high-intensity and weak signals in the same field of view.